Proteins and especially immunoglobulins play an important role in today's medical portfolio. Polypeptides for use in pharmaceutical applications are mainly produced in mammalian cells such as CHO cells, NS0 cells, Sp2/0 cells, COS cells, HEK cells, BHK cells, PER.C6® cells, and the like.
Due to their chemical and physical properties, such as molecular weight and domain architecture including secondary modifications, the downstream processing of immunoglobulins is very complicated. For example, are not only for formulated drugs but also for intermediates in downstream processing (DSP) concentrated solutions required to achieve low volumes for economic handling and application storage. The down stream processing of biotechnologically produced immunoglobulins in general comprises three chromatography steps: a first affinity chromatography step using e.g. Protein A, to remove non-immunoglobulin molecules, normally followed by two ion exchange chromatography steps, whereof the last step is a so called polishing step to remove DNA and HCP contaminants. The purified immunoglobulin is obtained in a low concentration solution requiring a concentration step prior to formulating the antibody into the pharmaceutical formulation. Due to the non-natural conditions required during the down stream processing the normally monomeric immunoglobulin tends to form dimers, oligomer and higher order aggregates. These aggregates do not possess the intended antigen-binding activity of the monomeric immunoglobulin and have to be removed. Ghose, S., et al. (Biotechnol. Bioeng. 87 (2004) 413-423) report preparative protein purification on underivatized silica. Reifsnyder, D. H., et al. (J. Chrom. A 753 (1996) 73-80) report the capture of IGF-I from a crude fermentation broth and a specific elution using a combination of ethanol and NaCl. Lifsics, M. R. and Williams, R. C. Jr (Biochem. 23 (1984) 2866-2875) report a molecular sieve chromatography in 8 M urea on controlled-pore glass for separating monomeric and aggregated forms of a protein from bovine neurofilaments. Ghose, S., et al. (Abstracts of Papers, 224th ACS National Meeting, Boston, Mass., United States, Aug. 18-22, 2002 (2002), BIOT-317 Publisher: American Chemical Society, Washington, D.C.) report the use of underivatized naked silica gel as a preparative stationary phase for process purification of proteins.
In U.S. Pat. No. 4,606,825 a method of separating and recovering immunoglobulin G using controlled pore glass bearing non-cross-linked covalently bound polyethylene imine functions is reported. A method for separating a polypeptide monomer from a mixture comprising dimers and/or multimers using an ion exchange chromatography resin and a gradient elution is reported in US 2002/0010319. Mizutani, T. and Mizutani, A., J. Chrom. 168 (1979) 143-150 report the comparison of elution patterns of proteins chromatographed on controlled-pore glass and carboxymethyl cellulose. The isolation and purification of the enzyme myeloperoxidase using a chromatography with carboxymethylated controlled pore glass is reported in DE 39 07 162 A1.